SUMMARY Bone diseases affect over 53 million people in United States. Low bone density and high bone density are two malfunctions that lead to osteoporosis and osteopetrosis, respectively. The last one is a rare disease affecting only 1250 adults in USA. Bone resorption relies on the recruitment of the Vacuolar H+-ATPases (V-ATPases) of osteoclasts for the acidification of the local extracellular environment to dissolve bone minerals. V-ATPases, membrane proton pumps, are highly conserved from yeast to humans and could serve as an effective therapeutic approach to reverse V-ATPase malfunction. V-ATPase is a multi-subunit molecular complex, where inter-subunit interactions are important for its function. Therefore, we hypothesize that by unraveling the V-ATPase's mechanism of action and identifying potential inhibitor binding sites, we could target bone resorption related diseases by structure-guided drug design. Using the yeast V-ATPase as a model system, we will identify the mechanism of action of known V-ATPases inhibitors: bafilomycin and concanamycin and luteolin. Currently, the mechanism of inhibition of these molecules is largely unknown mainly due to the lack of structural information of V-ATPase, which hinders de novo drug development efforts. Recently, the atomistic views of the yeast V-ATPase at three different functional states have been solved. To complement the published structural information of the yeast V-ATPase, the PI and his team propose to apply a hybrid approach composed of state-of-the-art fluorescence spectroscopic methods at a single molecular level in combination with computational modeling to determine inter-subunit interactions in the yeast V-ATPase to uncover the mechanism of inhibition of the mentioned inhibitors. The long-term goals is to help develop small- molecules that specifically target osteoclast specific V-ATPase as a direct therapeutic approach against bone diseases. With a track record in single molecule experiments delineating the structure-function relationship of large molecular complexes and membrane receptors, the PI and his research team are uniquely positioned to pursue the following specific aims: (1) Elucidate the interdomain motions between different subunits of the V- ATPase during the ATP hydrolysis cycle; (2) Determine the binding sites of existing small molecule effector; and (3) Determine the inhibition mechanism of existing small molecule effectors. The proposed project will use the computational and simulation COBRE CORE B to build structural models based on a hybrid experimental and computational approach. In addition, this project will use the microfabrication CORE C to develop custom- built microfluidic devices for studying the kinetic response of the V-ATPase at a single molecular level. Successful completion of the research project will support the PI and his team to establish the proof of concept as they transition into the study of the structure, dynamics and functions of human osteoclast-specific V- ATPase; thus, paving the molecular foundation for structure-based drug design to find novel potent inhibitors or promoters targeting various bone related diseases.